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Background & Aims To provide an adequate treatment strategy for chronic hepatitis B, it is essential to know which patients are expected to have a good prognosis and which patients do not require therapeutic intervention. Previously, we identified the substitution of isoleucine to leucine at amino acid 97 (I97L) in the hepatitis B core region as a key predictor among patients with stable hepatitis. In this study, we attempted to identify the point at which I97L affects the hepatitis B virus (HBV) life cycle and to elucidate the underlying mechanisms governing the stabilization of hepatitis. Methods To confirm the clinical features of I97L, we used a cohort of hepatitis B e antigen–negative patients with chronic hepatitis B infected with HBV-I97 wild-type (wt) or HBV-I97L. The effects of I97L on viral characteristics were evaluated by in vitro HBV production and infection systems with the HBV reporter virus and cell culture-generated HBV. Results The ratios of reduction in hepatitis B surface antigen and HBV DNA were higher in patients with HBV-I97L than in those with HBV-I97wt. HBV-I97L exhibited lower infectivity than HBV-I97wt in both infection systems with reporter HBV and cell culture-generated HBV. HBV-I97L virions exhibiting low infectivity primarily contained a single-stranded HBV genome. The lower efficiency of cccDNA synthesis was demonstrated after infection of HBV-I97L or transfection of the molecular clone of HBV-I97L. Conclusions The I97L substitution reduces the level of cccDNA through the generation of immature virions with single-stranded genomes. This I97L-associated low efficiency of cccDNA synthesis may be involved in the stabilization of hepatitis., Graphical abstract

Hepatitis B virus (HBV) infection is one of the most serious global health problems. Our previous data using an in vitro assay revealed that miR-6126 suppressed the extracellular HBs antigen level, suggesting that miR-6126 had potential to suppress viral activity of HBV. In the current study, we aimed to clarify whether miR-6126 downregulated the expression level of sodium taurocholate cotransporting polypeptide (NTCP), a host cell receptor required for HBV entry. In brief, HepG2-NTCP cells were utilized to evaluate the expression level of NTCP and the PreS1 attachment to NTCP after transfection with miR-6126. The protein expression level of NTCP was evaluated using Western blot analysis and immunostaining. In addition to HepG2-NTCP cells, PXB cells were also utilized to validate inhibitory effect of miR-6126 on PreS1 attachment. The HBs antigen level in the culture supernatant was measured to evaluate reduction of HBV entry into hepatocytes. The stability of NTCP mRNA was evaluated to ascertain the cause of the downregulation of NTCP mRNA. The expression profile of messenger RNAs was evaluated using next-generation sequencing to search for direct targets of miR-6126. Consequently, transfection of miR-6126 decreased the NTCP expression level in HepG2-NTCP cells. Attachment of the PreS1 probe on the cell surface decreased in HepG2-NTCP cells and PXB cells, primary human hepatocytes. HBs antigen level in the culture supernatant also declined in PXB cells. Stability of NTCP mRNA was reduced by miR-6126 transfection in HepG2 cells. In conclusion, miR-6126 downregulated the expression of NTCP mRNA, which contributed to the inhibition of HBV entry into hepatocytes exerted by miR-6126.

Hepatitis B virus (HBV) infection is a major public health problem. The sodium taurocholate cotransporting polypeptide (NTCP) has been identified as an essential HBV receptor. Human hepatocytes are infected with HBV via binding between the preS1 region of the HBV large envelope protein and the NTCP. However, the role of preS2 in HBV entry is not well understood. In this study, we induced anti-preS2 serum in mice by DNA immunization, and showed that the resulting antiserum neutralized HBV infectivity. Competition assays using overlapping peptides suggested that the neutralizing epitope is located in the N-terminal region of preS2. In addition, monoclonal antibodies targeting the N-terminal region of preS2 neutralized HBV infectivity, indicating that these domains are critical epitopes for viral neutralization. These findings provide new insights into the HBV entry machinery while suggesting a novel modality for the prevention and treatment of HBV infection.

Hepatitis B virus (HBV) infects 240 million people worldwide. Current therapy profoundly suppresses HBV replication but requires long-term maintenance therapy. Therefore, there is still a medical need for an efficient HBV cure. HBV enters host cells by binding via the preS1 domain of the viral L protein to the Na

Hepatitis B virus (HBV) infection, which increases the risk of cirrhosis and hepatocellular carcinoma and requires lifelong treatment, has become a major global health problem. However, host factors essential to the HBV life cycle are still unclear, and the development of new drugs is needed. Cells derived from the human hepatoma cell line HepG2 and engineered to overexpress sodium taurocholate cotransporting polypeptide (NTCP: a receptor for HBV), termed HepG2/NTCP cells, are widely used as the cell-based HBV infection and replication systems for HBV research. We recently found that human hepatoma cell line Li23-derived cells overexpressing NTCP (A8 cells subcloned from Li23 cells), whose gene expression profile was distinct from that of HepG2/NTCP cells, were also sensitive to HBV infection. However, the HBV susceptibility of A8 cells was around 1/100 that of HepG2/NTCP cells. Since we considered that plural cell assay systems will be needed for the objective evaluation of anti-HBV reagents, as we previously demonstrated in hepatitis C virus research, we here attempted to develop a new Li23 cell-derived assay system equivalent to that using HepG2/NTCP cells. By repeated subcloning of A8 cells, we successfully established a new cell line (A8.15.78.10) exhibiting high HBV susceptibility equal to that of HepG2/NTCP cells. Characterization of A8.15.78.10 cells revealed that the increase of HBV susceptibility was correlated with increases in the protein and glycosylation levels of NTCP, and with decreased expression of STING, a factor contributing to innate immunity. Finally, we performed a comparative evaluation of HBV entry inhibitors (cyclosporin A and rosiglitazone) by an HBV/secNL reporter assay using A8.15.78.10 cells or HepG2/NTCP cells. The results confirmed that cyclosporin A exhibited anti-HBV activity in both cell lines, as previously reported. However, we found that rosiglitazone did not show the anti-HBV activity in A8.15.78.10 cells, although it worked in HepG2/NTCP cells as previously reported. This suggested that the difference in anti-HBV activity between cyclosporin A and rosiglitazone was due to the different types of cells used for the assay. In conclusion, plural assay systems using different types of cells are required for the objective and impartial evaluation of anti-HBV reagents.

Hepatitis B virus (HBV) infection is a major public health problem. Human hepatocytes are infected with HBV via binding between the preS1 region in the large envelope protein of HBV and sodium taurocholate cotransporting polypeptide. Although several monoclonal antibodies (MAbs) that recognize the receptor binding domain in preS1 and neutralize HBV infection have been isolated, details of neutralizing epitopes are not understood. In this study, we generated 13 MAbs targeting the preS1 receptor binding domain from preS1-specific memory B cells derived from DNA-immunized mice. The MAbs were classified into three groups according to the epitope regions, designated epitopes I to III. A virus neutralization assay revealed that MAbs recognizing epitopes I and III neutralized HBV infection, suggesting that these domains are critical epitopes for viral neutralization. In addition, a neutralization assay against multiple genotypes of HBV revealed that epitope I is a semipangenotypic neutralizing epitope, whereas epitope III is a genotype-specific epitope. We also showed that neutralizing MAbs against preS1 could neutralize HBV bearing a vaccine-induced escape mutation. These findings provide insight into novel immunoprophylaxis for the prevention and treatment of HBV infection. IMPORTANCE The HBV preS1 amino acid 2 to 47 region (preS1/2–47) is essential for virus binding to sodium taurocholate cotransporting polypeptide. Several MAbs targeting preS1/2–47 have been reported to neutralize HBV infection; however, which region in preS1/2–47 contains the critical neutralizing epitope(s) for HBV infection is unclear. Here, we generated several MAbs targeting preS1/2–47, and we found that MAbs recognizing the N or C terminus of preS1/2–47 remarkably neutralized HBV infection. We further confirmed the neutralizing activity of anti-preS1 MAbs against HBV with a vaccine escape mutation. These data clarified the relationship between the antibody epitope and the virus-neutralizing activity and also suggested the potential ability of a vaccine antigen containing the preS1 region to overcome the weakness of current hepatitis B vaccines comprising the small S protein.

Hepatocytes derived from human iPSCs are useful to study hepatitis B virus (HBV) infection, however infection efficiency is rather poor. In order to improve the efficiency of HBV infection to iPSC-derived hepatocytes, we set a co-culture of hepatocytes with liver non-parenchymal cells and found that liver sinusoidal endothelial cells (LSECs) enhanced HBV infection by secreting epidermal growth factor (EGF). While EGF receptor (EGFR) is known as a co-receptor for HBV, we found that EGF enhanced HBV infection at a low dose of EGF, whereas EGF at a high dose suppressed HBV infection. EGFR is internalized by clathrin-mediated endocytosis (CME) and clathrin-independent endocytosis (CIE) pathways depending on the dose of EGF. At a high dose of EGF, the endocytosed EGFR via CIE is degraded in the lysosome. This study is the first to provide evidence that HBV is endocytosed via CME and CIE pathways at a low and high dose of EGF, respectively. In conclusion, we developed an in vitro system of HBV infection using iPSC-derived liver cells, and show that EGF secreted from LSECs modulates HBV infection in a dose dependent manner.

Hepatitis B virus (HBV), a highly persistent pathogen causing hepatocellular carcinoma (HCC), takes full advantage of host machinery, presenting therapeutic targets. Here we aimed to identify novel druggable host cellular factors using the reporter HBV we have recently generated. In an RNAi screen of G protein-coupled receptors (GPCRs), GPCR39 (GPR39) appeared as the top hit to facilitate HBV proliferation. Lentiviral overexpression of active GPR39 proteins and an agonist enhanced HBV replication and transcriptional activities of viral promoters, inducing the expression of CCAAT/enhancer binding protein (CEBP)-&beta, (CEBPB). Meanwhile, GPR39 was uncovered to activate the heat shock response, upregulating the expression of proviral heat shock proteins (HSPs). In addition, glioma-associated oncogene homologue signaling, a recently reported target of GPR39, was suggested to inhibit HBV replication and eventually suppress expression of CEBPB and HSPs. Thus, GPR39 provirally governed intracellular circuits simultaneously affecting the carcinopathogenetic gene functions. GPR39 and the regulated signaling networks would serve as antiviral targets, and strategies with selective inhibitors of GPR39 functions can develop host-targeted antiviral therapies preventing HCC.

Summary SynNotch receptor technology is a versatile tool that uses the regulatory notch core portion with an extracellular scFv and an intracellular transcription factor that enables to program customized input and output functions in mammalian cells. In this study, we designed a novel synNotch receptor comprising scFv against HBs antigen linked with an intracellular artificial transcription factor and exploited it for viral sensing and cellular immunotherapy. The synNotch receptor expressing cells sensed HBV particles and membrane-bound HBs antigens and responded by expressing reporter molecules, secNL or GFP. We also programmed these cells to dispense antiviral responses such as type I interferon and anti-HBV neutralizing mouse-human chimeric antibodies. Our data reveal that synNotch receptor signaling works for membrane-bound ligands such as enveloped viral particles and proteins borne on liposomal vesicles. This study establishes the concepts of “engineered immunity” where the synNotch platform is utilized for cellular immunotherapy against viral infections., Graphical Abstract, Highlights • We designed synNotch receptor with scFv to sense HBs antigen • SynNotch-receptor-transduced cells can express reporter and antiviral molecules • Membrane-bound viral surface proteins can activate synNotch signaling • SynNotch technology can foster “engineered immunity” against viral infections, Immunity; Viral Microbiology; Bioengineering

Chronic infection with Hepatitis B virus (HBV) increases the risk of developing fibrosis, cirrhosis, or hepatocellular carcinoma. Current therapies are limited to type-I interferons and/or nucleos(t)ide analogues; however, these are only partially effective. The development of novel anti-HBV agents for new treatment strategies has been hampered by the lack of a suitable system that allows the in vitro replication of HBV. Studies of virus infection/replication at the molecular level using wild-type HBV are labor-intensive and time-consuming. To overcome these problems, we previously constructed a recombinant reporter HBV bearing the NanoLuc gene and showed its usefulness to identify factors that affect HBV proliferation. Because this system mimics the early stage of HBV life cycle faithfully, we conducted a quantitative analysis of HBV infectivity to several human hepatocyte cell lines as well as the effect of dimethyl sulfoxide and HBV protein X on the early stage of HBV proliferation using this system. Furthermore, we developed a system to produce a reporter HBV expressing a pol gene. These reporter HBVs may provide an opportunity to enhance our understanding of the HBV life cycle and aid strategies for the development of new anti-HBV agents. This article is protected by copyright. All rights reserved.

Antiviral therapies for chronic hepatitis B virus (HBV) infection that are currently applicable for clinical use are limited to nucleos(t)ide analogs targeting HBV polymerase activity and pegylated interferon alpha (PEG-IFN). Towards establishing an effective therapy for HBV related diseases, it is important to develop a new anti-HBV agent that suppresses and eradicates HBV. This study used recombinant HBV encoding NanoLuc to screen anti-HBV compounds from 1827 US Food and Drug Administration approved compounds and identified several compounds that suppressed HBV infection. Among them, KX2-391, a non-ATP-competitive inhibitor of SRC kinase and tubulin polymerization, was identified as a lead candidate for an anti-HBV drug. Treatment of sodium taurocholate cotransporting polypeptide (NTCP) transduced-HepG2 (HepG2-NTCP) or primary human hepatocytes with KX2-391 suppressed HBV replication in a dose-dependent manner. The anti-HBV activity of KX2-391 appeared not to depend on SRC kinase activity because siRNA for SRC mRNA did not impair the HBV infection/replication. The anti-HBV activity of KX2-391 depended on the inhibitory effect of tubulin polymerization similar to other tubulin polymerization inhibitors, some of which were shown to inhibit HBV replication. KX2-391 inhibited HBV transcription driven by a HBV precore promoter in an HBV X protein-independent manner but did not inhibit the activity of HBV-S1, -S2, -X or cytomegalovirus promoters. Treatment with KX2-391 reduced the expression of several various factors including hepatocyte nuclear factor-4a.

Aim Interferon (IFN)-λ3 is known to have antiviral effects against various pathogens. Recently, it has been reported that the production of IFN-λ3 in colon cells after the administration of nucleotide analogs is expected to reduce hepatitis B surface antigen in chronic hepatitis B patients. Here, we aimed to prove the antiviral effects of IFN-λ3 on hepatitis B virus (HBV) by using an in vitro HBV production and infection system. Methods We used HepG2.2.15-derived HBV as an inoculum and the replication-competent molecular clone of HBV as a replication model. Results By administering IFN-λ3 to HepG2 cells transfected with the HBV molecular clone, the production of hepatitis B surface antigen and hepatitis B core-related antigen was reduced dose-dependently. IFN-λ3 treatment also reduced the number of HBV-positive cells and the synthesis of covalently closed circular DNA after infection of HepG2.2.15-derived HBV to sodium taurocholate cotransporting polypeptide-transduced HepG2 cells. The inhibitory effect on HBV infection by IFN-λ3 was confirmed by using a recombinant a HBV reporter virus system. To elucidate the underlying mechanisms of the anti-HBV effect of IFN-λ3, we assessed the transcription of HBV RNA and the production of core-associated HBV DNA in HBV molecular clone-transfected HepG2 cells, and found that both parameters were reduced by IFN-λ3. Conclusions We observed that the administration of IFN-λ3 inhibits HBV infection and the production of HBV proteins at the HBV RNA transcription level. This finding provides novel insight into the treatment of chronic hepatitis B patients with the administration or induction of IFN-λ3.

Here, we identify ATP1B3 and fibrillin-1 as novel BST-2-binding proteins. ATP1B3 depletion in HeLa cells (BST-2-positive cells), but not 293T cells (BST-2-negative cells), induced the restriction of HIV-1 production in a BST-2-dependent manner. In contrast, fibrillin-1 knockdown reduced HIV-1 production in 293T and HeLa cells in a BST-2-independent manner. Moreover, NF-κB activation was enhanced by siATP1B3 treatment in HIV-1- and HIV-1ΔVpu-infected HeLa cells. In addition, ATP1B3 silencing induced high level BST-2 expression on the surface of HeLa cells. These results indicate that ATP1B3 is a co-factor that accelerates BST-2 degradation and reduces BST-2-mediated restriction of HIV-1 production and NF-κB activation.

A recombinant hepatitis B virus (HBV) expressing NanoLuc (NL) (HBV/NL) was produced by cotransfecting a plasmid containing a 1.2-fold HBV genome carrying the NL gene with a plasmid bearing a packaging-defective 1.2-fold HBV genome into a human hepatoma cell line, HepG2. We found that NL activity in HBV/NL-infected primary hepatocytes or sodium taurocholate cotransporting polypeptide-transduced human hepatocyte-derived cell lines increased linearly for several days after infection and was concordant with HBV RNA levels in the cells. Treatment of the virus-infected cells with HBV inhibitors reduced NL activity in a dose-dependent manner. Detection of HBV/NL infection, monitored by NL activity, was highly sensitive and less expensive than detection using the conventional method to evaluate HBV infection. In addition, because we also studied host factors, this system is applicable not only for studying the HBV life cycle, but also for exploring agent(s) that regulate HBV proliferation.

The therapeutic goal for hepatitis B virus (HBV) infection is HBs antigen (HBsAg) seroclearance, which is achieved through 48-week pegylated interferon (Peg-IFN) therapy. This study aimed to identify predictive biomarkers for sustained HBsAg reduction by analyzing serum microRNAs. Twenty-two consecutive chronic HBV infection patients negative for HBe antigen (HBeAg) with HBV-DNA levels &lt, 5 log copies/mL, alanine aminotransferase (ALT) &lt, 100 U/L, and compensated liver functions, were enrolled. The patients were subcutaneously injected with Peg-IFN&alpha, 2a weekly for 48 weeks (treatment period), followed by the 48-week observation period. HBsAg 1-log drop relative to baseline levels recorded at the end of the observation period was considered effective. Sera were obtained at weeks 0 and 24 during the treatment period analyzed for microRNAs. The microRNA (miRNA) antiviral activity was evaluated in vitro using Huh7/sodium taurocholate cotransporting polypeptide (NTCP) cells. As a result, six patients achieved the HBsAg 1-log drop after the observation periods. Comparison of serum microRNA levels demonstrated that high miR-6126 levels at week 24 predicted HBsAg 1-log drop. Furthermore, miR-6126 reduced HBsAg in culture medium supernatants and intracellular HBV-DNA quantities in Huh7/NTCP cells. In conclusion, high serum miR-6126 levels during Peg-IFN therapy predicted the HBsAg 1-log drop 48 weeks after the completion of therapy. In vitro assays revealed that miR-6126 was able to suppress HBsAg production and HBV replication.

Sodium taurocholate cotransporting polypeptide (NTCP) is a major entry receptor of hepatitis B virus (HBV) and one of the most attractive targets for anti-HBV drugs. We developed a cell-mediated drug screening method to monitor NTCP expression on the cell surface by generating a HepG2 cell line with tetracycline-inducible expression of NTCP and a monoclonal antibody that specifically detects cell-surface NTCP. Using this system, we screened a small molecule library for compounds that protected against HBV infection by targeting NTCP. We found that glabridin, a licorice-derived isoflavane, could suppress viral infection by inducing caveolar endocytosis of cell-surface NTCP with an IC50 of ~40 μM. We also found that glabridin could attenuate the inhibitory effect of taurocholate on type I interferon signaling by depleting the level of cell-surface NTCP. These results demonstrate that our screening system could be a powerful tool for discovering drugs targeting HBV entry.

Hepatitis B virus (HBV) is a global major health problem, with over one million deaths annually caused by chronic liver damage. Understanding host factors that modulate HBV replication may aid the development of anti-HBV therapies. Our recent genome-wide small interfering RNA screen using recombinant HBV demonstrated that TIP60 inhibited HBV infection. Here, we show that TIP60 complex contributes to anti-HBV defense. The TIP60 complex bound to the HBV promoter and suppressed HBV transcription driven by the precore/core promoter. The silencing of EP400, TRRAP, BAF53a, RUVBL1, and RUVBL2, which form the TIP60 complex, also resulted in increased HBV transcription. These results contribute to our enhanced understanding of the molecular mechanism of HBV transcription associated with the chromatin structure of HBV covalently closed circular DNA (cccDNA). Exploiting these intrinsic cellular defenses might help develop new anti-HBV agents. IMPORTANCE Investigating the molecular mechanism of HBV replication is important to understand the persistent nature of HBV infection and to aid the development of new HBV agents, which are currently limited to HBV polymerase inhibitors. Previously, we developed a new reporter HBV. By screening host factors using this recombinant virus, we identified several gene products that regulate HBV infection, including TIP60. Here, we showed that TIP60, a catalytic subunit of the NuA4 complex, inhibited HBV replication. Depletion of TIP60 increased the level of HBV mRNA. Moreover, TIP60 localized in the HBV cccDNA chromatin complex catalyzed the acetylation of histone H4 to recruit Brd4. These results suggest that TIP60, in concert with other cellular factors, plays an important role in the regulation of the HBV chromatin structure by acting as a critical component of the intrinsic antiviral defense, which sheds new light on the regulation of HBV replication.

Hepatitis B virus (HBV) is the major cause of liver cirrhosis and hepatocellular carcinoma. After entering a hepatocyte, HBV forms a nuclear viral episome and produces pregenomic (pg) RNA with a stem-loop structure called an epsilon, which acts to signal encapsidation. We previously demonstrated that TGF-β upregulates activation-induced cytidine deaminase (AID) expression in hepatocytes, which in turn downregulates HBV transcripts by recruiting the RNA exosome complex. The molecular mechanism underlying AID-mediated HBV RNA reduction remains largely unclear. Here we used a pgRNA reporter system having a reporter gene within pgRNA to identify sis- and trans-acting elements in AID-mediated HBV RNA reduction. We found that the epsilon RNA and C-terminus of AID are required for AID-mediated HBV RNA reduction. Importantly, this reduction was reproduced in a hydrodynamic HBV transfection mouse model. The molecular mechanism of AID-mediated HBV RNA reduction is discussed.

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), that carry foreign genes such as a reporter or marker protein in their genomes. These recombinant viruses usually faithfully mimic the life cycle of the original virus in infected cells and exhibit the same host range dependence. The development of a recombinant virus enables the efficient screening of inhibitors and the identification of specific host factors. However, to date the construction of recombinant hepatitis B virus (HBV) has been difficult because of various experimental limitations. The main limitation is the compact genome size of HBV, and a fairly strict genome size that does not exceed 1.3 genome sizes, that must be packaged into virions. Thus, the size of a foreign gene to be inserted should be smaller than 0.4 kb if no deletion of the genome DNA is to be performed. Therefore, to overcome this size limitation, the deletion of some HBV DNA is required. Here, we report the construction of recombinant HBV encoding a reporter gene to monitor the early stage of the HBV replication cycle by replacing part of the HBV core-coding region with the reporter gene by deleting part of the HBV pol coding region. Detection of recombinant HBV infection, monitored by the reporter activity, was highly sensitive and less expensive than detection using the currently available conventional methods to evaluate HBV infection. This system will be useful for a number of applications including high-throughput screening for the identification of anti-HBV inhibitors, host factors and virus-susceptible cells.

Apolipoprotein B mRNA-editing enzyme, catalytic polypeptide 1 (APOBEC1) is an intestine-specific RNA-binding protein. However, inflammation or exposure to DNA-damaging agents can induce ectopic APOBEC1 expression, which can result in hepatocellular hyperplasia in animal models. To identify its RNA targets, FLAG-tagged APOBEC1 was immunoprecipitated from transfected HuH7.5 hepatocellular carcinoma cells and analyzed using DNA microarrays. The interleukin-8 (IL8) mRNA was the most abundant co-precipitated RNA. Exogenous APOBEC1 expression increased IL8 production by extending the half-life of the IL8 mRNA. A cluster of AU-rich elements in the 3'-UTR of IL8 was essential to the APOBEC1-mediated increase in IL8 production. Notably, IL8 mRNA did not co-immunoprecipitate with APOBEC1 from lysates of other cell types at appreciable levels; therefore, other factors may enhance the association between APOBEC1 and IL8 mRNA in a cell type-specific manner. A yeast two-hybrid analysis and siRNA screen were used to identify proteins that enhance the interaction between APOBEC1 and IL8 mRNA. Heterogeneous nuclear ribonucleoprotein Q (hnRNPQ) was essential to the APOBEC1/IL8 mRNA association in HuH7.5 cells. Of the seven hnRNPQ isoforms, only hnRNPQ6 enabled APOBEC1 to bind to IL8 mRNA when overexpressed in HEK293 cells, which expressed the lowest level of endogenous hnRNPQ6 among the cell types examined. The results of a reporter assay using a luciferase gene fused to the IL8 3'-UTR were consistent with the hypothesis that hnRNPQ6 is required for APOBEC1-enhanced IL8 production. Collectively, these data indicate that hnRNPQ6 promotes the interaction of APOBEC1 with IL8 mRNA and the subsequent increase in IL8 production.

Previous studies have demonstrated that cyclopentenone prostaglandins (cyPGs) inhibit human immunodeficiency virus type 1 (HIV-1) replication in various cell types. This antiviral activity has been associated with the induction of heat-shock protein 70 (HSP70) in infected cells. We investigated a new role of prostaglandin A 1 (PGA 1 ) in the replication of HIV-1 in non-permissive cells. Because overexpression of HSP70 blocks the viral infectivity factor (Vif)-mediated degradation of APOBEC3G (A3G) via the ubiquitin–proteasome pathway, we examined the effects of PGA 1 on A3G and HIV-1 replication. The induction of HSP70 synthesis by PGA 1 blocked Vif-mediated A3G degradation and enhanced the incorporation of A3G into both wild-type and Vif-deficient viruses. Furthermore, we determined the viral titer of HIV-1 particles produced from PGA 1 -treated 293T cells. The induction of HSP70 synthesis by PGA 1 significantly reduced the viral titer in the presence of A3G. Additionally, the p24 Gag antigen levels were dramatically reduced in non-permissive cells treated once or repeatedly with PGA 1 . Thus, we showed that PGA 1 inhibits HIV-1 replication, at least in part, by blocking Vif-mediated A3G degradation.

Inflammatory cytokines and chemokines play important roles in inflammation during viral infection. Hepatitis C virus (HCV) is a hepatotropic RNA virus that is closely associated with chronic liver inflammation, fibrosis, and hepatocellular carcinoma. During the progression of HCV-related diseases, hepatic stellate cells (HSCs) contribute to the inflammatory response triggered by HCV infection. However, the underlying molecular mechanisms that mediate HSC-induced chronic inflammation during HCV infection are not fully understood. By coculturing HSCs with HCV-infected hepatocytes in vitro , we found that HSCs stimulated HCV-infected hepatocytes, leading to the expression of proinflammatory cytokines and chemokines such as interleukin-6 (IL-6), IL-8, macrophage inflammatory protein 1α (MIP-1α), and MIP-1β. Moreover, we found that this effect was mediated by IL-1α, which was secreted by HSCs. HCV infection enhanced production of CCAAT/enhancer binding protein (C/EBP) β mRNA, and HSC-dependent IL-1α production contributed to the stimulation of C/EBPβ target cytokines and chemokines in HCV-infected hepatocytes. Consistent with this result, knockdown of mRNA for C/EBPβ in HCV-infected hepatocytes resulted in decreased production of cytokines and chemokines after the addition of HSC conditioned medium. Induction of cytokines and chemokines in hepatocytes by the HSC conditioned medium required a yet to be identified postentry event during productive HCV infection. The cross talk between HSCs and HCV-infected hepatocytes is a key feature of inflammation-mediated, HCV-related diseases.

Despite the breadth of knowledge that exists regarding the function of long noncoding RNAs (lncRNAs) in biological phenomena, the role of lncRNAs in host antiviral responses is poorly understood. Here, we report that lncRNA#32 is associated with type I IFN signaling. The silencing of lncRNA#32 dramatically reduced the level of IFN-stimulated gene (ISG) expression, resulting in sensitivity to encephalomyocarditis virus (EMCV) infection. In contrast, the ectopic expression of lncRNA#32 significantly suppressed EMCV replication, suggesting that lncRNA#32 positively regulates the host antiviral response. We further demonstrated the suppressive function of lncRNA#32 in hepatitis B virus and hepatitis C virus infection. lncRNA#32 bound to activating transcription factor 2 (ATF2) and regulated ISG expression. Our results reveal a role for lncRNA#32 in host antiviral responses.

Hepatitis B virus (HBV) is not eradicated by current antiviral therapies due to persistence of HBV covalently closed circular DNA (cccDNA) in host cells and thus development of novel culture models for productive HBV infection is urgently needed, which will allow the study of HBV cccDNA eradication. To meet this need, we developed culture models of HBV infection using human induced pluripotent stem cell-derived hepatocyte lineages, including immature proliferating hepatic progenitor-like cell lines (iPS-HPCs) and differentiated hepatocyte-like cells (iPS-Heps). These cells were susceptible to HBV infection, produced HBV particles and maintained innate immune responses. The infection efficiency of HBV in iPS-HPCs predominantly depended on the expression levels of sodium taurocholate cotransporting polypeptide (NTCP) and was low relative to iPS-Heps: however, long-term culture of iPS-Heps was difficult. To provide a model for HBV persistence, iPS-HPCs overexpressing NTCP were established. The long-term persistence of HBV cccDNA was detected in iPS-HPCs overexpressing NTCP and depended on the inhibition of the Janus-kinase signaling pathway. In conclusion, this study provides evidence that iPS-derived hepatic cell lines can be utilized for novel HBV culture models with genetic variation to investigate the interactions between HBV and host cells and the development of anti-HBV strategies.

Persistent hepatitis C virus (HCV) infection is a leading cause of liver diseases, including chronic hepatitis C, progressive liver fibrosis, cirrhosis, and hepatocellular carcinoma. Chronic hepatitis C is strongly associated with lipid accumulation in the hepatocytes. A significant percentage of chronic HCV patients suffer from diabetes and steatosis. Accumulating evidence indicates a strong relationship between lipid metabolism and HCV proliferation. HCV contains lipoprotein components in its envelope, a unique characteristic that results in the lower buoyant density of HCV virions than those of other enveloped viruses. The incorporation of lipoprotein components is required for viral infection. During the egress of HCV, host factors, such as microsomal triglyceride transfer protein, play some role in the association between lipoprotein and a precursor form of HCV. In this chapter, we summarize how HCV uses the processes of lipid metabolism and discuss the importance of lipoprotein components in the viral lifecycle.

Various host factors are involved in the cellular entry of hepatitis C virus (HCV). In addition to the factors previously reported, we discovered that the very-low-density lipoprotein receptor (VLDLR) mediates HCV entry independent of CD81. Culturing Huh7.5 cells under hypoxic conditions significantly increased HCV entry as a result of the expression of VLDLR, which was not expressed under normoxic conditions in this cell line. Ectopic VLDLR expression conferred susceptibility to HCV entry of CD81-deficient Huh7.5 cells. Additionally, VLDLR-mediated HCV entry was not affected by the knockdown of cellular factors known to act as HCV receptors or HCV entry factors. Because VLDLR is expressed in primary human hepatocytes, our results suggest that VLDLR functions in vivo as an HCV receptor independent of canonical CD81-mediated HCV entry.

Pattern recognition receptors (PRRs) play a pivotal role in host innate immune responses against microbial infection. Viruses are primarily recognized by PRRs such as Toll-like receptor 3, 7, 8 and 9, and RIG-I-like receptors. Recent studies have demonstrated that DNA-dependent activator of IFN-regulatory factors (DAI) is a cytosolic sensor molecule for dsDNA, and is implicated in antiviral responses to some DNA viruses. Soon after infection, human immunodeficiency virus type-1 (HIV-1) synthesizes viral dsDNA in the cytoplasm by reverse transcriptase. In addition, an immune compromised state due to chronic HIV-1 infection results in opportunistic infection with some microbes that potentially activate DAI. However, it has not been elucidated whether DAI affects HIV-1 replication, or its possible mechanisms. Here, we showed that forced expression of DAI markedly enhanced HIV-1 replication, which was largely impaired by mutations at κB sites in HIV-1 LTR or by suppressing activation of NF-κB. Moreover, intact structure around the D3 region (174-232 aa) and two RIP homotypic interaction motifs (198-214 aa and 256-272 aa) within DAI were critical for its activity. These results suggest that activation of DAI might contribute to augment HIV-1 replication through DAI- NF-κB pathway.

Double-stranded RNAs suppress the expression of homologous genes through an evolutionarily conserved process called RNA interference (RNAi) or post-transcriptional gene silencing. A bidentate nuclease called Dicer has been implicated as the protein responsible for the production of short interfering RNAs (siRNAs). In our experiments, Rex overexpression reduced the efficiency of short hairpin RNA (shRNA)-mediated RNAi. The interaction of Dicer with Rex inhibited the conversion of shRNA to siRNA. These results suggest that the interaction of Dicer with HTLV-I Rex inhibits Dicer activity and thereby reduces the efficiency of the conversion of shRNA to siRNA.Structured summaryMINT-7996954: Rex (uniprotkb:P0C205) physically interacts (MI:0915) with Dicer (uniprotkb:Q9UPY3) by pull down (MI:0096)MINT-7996926: Rex (uniprotkb:P0C205) and Dicer (uniprotkb:Q9UPY3) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT-7996905: Dicer (uniprotkb:Q9UPY3) physically interacts (MI:0915) with Rex (uniprotkb:P0C205) by anti bait coimmunoprecipitation (MI:0006)MINT-7996852, MINT-7996870, MINT-7996886, MINT-7996830: Rex (uniprotkb:P0C205) physically interacts (MI:0915) with Dicer (uniprotkb:Q9UPY3) by anti tag coimmunoprecipitation (MI:0007)

Severe acute respiratory syndrome coronavirus (SARS-CoV) causes a lung disease with high mortality. In addition, osteonecrosis and bone abnormalities with reduced bone density have been observed in patients following recovery from SARS, which were partly but not entirely explained by the short-term use of steroids. Here, we demonstrate that human monocytes, potential precursors of osteoclasts, partly express angiotensin converting enzyme 2 (ACE2), a cellular receptor of SARS-CoV, and that expression of an accessory protein of SARS-CoV, 3a/X1, in murine macrophage cell line RAW264.7 cells, enhanced NF-kappaB activity and differentiation into osteoclast-like cells in the presence of receptor activator of NF-kappaB ligand (RANKL). Furthermore, human epithelial A549 cells expressed ACE2, and expression of 3a/X1 in these cells up-regulated TNF-alpha, which is known to accelerate osteoclastogenesis. 3a/X1 also enhanced RANKL expression in mouse stromal ST2 cells. These findings indicate that SARS-CoV 3a/X1 might promote osteoclastogenesis by direct and indirect mechanisms.

Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia (ATL), HTLV-1-associated myelopathy/tropical spastic paraparesis, and other inflammatory diseases. Despite such severe outcomes of HTLV-1 infection, the level of HTLV-1 expression in vivo is very low and rapidly increases after transfer of cells to culture conditions. The mechanisms of this phenomenon have remained obscure. In the present study, we found that human and mouse stromal cells, such as epithelial cells and fibroblasts, suppressed HTLV-1 expression in ATL and non-ATL HTLV-1-infected cells. HTLV-1 mRNA and proteins in HTLV-1-infected cells markedly decreased upon coculture with human epithelial-like cells (HEK293T) or mouse embryo fibroblasts (NIH 3T3). When infected cells were reisolated from the cocultures, viral expression was restored to the original level over the following 48 h. Spontaneous induction of HTLV-1 expression in primary ATL cells in the first 24 h of culture was also inhibited by coculture with HEK293T cells. Coculture of HTLV-1-infected cells and HEK293T cells induced type I interferon responses, as detected by beta interferon (IFN-β) promoter activation and IFN-stimulated gene upregulation. HEK293T-mediated suppression of HTLV-1 expression was partly inhibited by antibodies to human IFN-α/β receptor. NIH 3T3-mediated suppression was markedly abrogated by neutralizing antibodies to mouse IFN-β. Furthermore, viral expression in HTLV-1-infected cells was significantly suppressed when the infected cells were intraperitoneally injected into wild-type mice but not IFN regulatory factor 7 knockout mice that are deficient of type I IFN responses. These findings indicate that the innate immune system suppresses HTLV-1 expression in vivo, at least through type I IFN.

Hepatitis C virus (HCV) is a major public health problem, as 170 million people worldwide are currently chronically infected with the virus. HCV infection leads to chronic inflammation, which is the initial step toward fibrosis and is a significant risk factor for developing hepatocellular carcinoma. HCV-induced liver inflammation involves several events, such as modification of cytokine and chemokine pathways, oxidative stress, and induction of steatosis. Recent studies have revealed that not only HCV-infected hepatocytes but also neighboring cells, such as lymphocytes, Kupffer cells and hepatic stellate cells (HSCs), play important roles in HCV-induced inflammation. In the current study, we found evidence of cross-talk between HCV-infected hepatocytes and HSCs, revealed by the production of cytokines and chemokines. Upon co-culture of HSCs with HCV-infected hepatocytes in vitro, HSCs stimulated HCV-infected hepatocytes to produce pro-inflammatory cytokines and chemokines, including interleukin (IL)-6, IL-8, macrophage inflammatory protein (MIP)-1α and MIP-1β. This cross-talk is likely to be a key feature of inflammatory diseases caused by HCV infection.

Gene targeting in mammalian cells through the use of short-hairpin RNAs (shRNAs) has been advanced by the development of vector systems for efficient delivery and stable expression of shRNA sequences (4, 7, 22, 30). Upon delivery into cells, shRNAs are converted into short double-stranded RNAs, termed small interfering RNAs (siRNAs), that mediate a sequence-specific RNA degradation process termed RNA interference (RNAi) (12, 14, 42). Antiviral therapy based on siRNA has been proposed as a new method for intracellular immunization against human immunodeficiency virus type 1 (HIV-1) (16, 31, 32) and hepatitis C virus (HCV) (34). When viral genes are targeted, viruses can escape from RNAi-mediated inhibition due to their high mutation rate (6, 11, 39). An alternative approach that shows promise is the use of siRNAs targeting cellular genes essential for virus replication. In the case of HIV-1, siRNAs against the cell surface CD4 receptor (31) or CXCR4 and CCR5 coreceptors for HIV-1 entry conferred viral resistance (2, 3, 33). However, CD4 and CXCR4 are essential for T-cell development and proper immunologic function. In addition, although CCR5 might be nonessential for normal function (23), not all HIV-1 strains require CCR5. Downregulation of an essential cellular coreceptor could potentially result in the emergence of HIV-1 variants that use another coreceptor(s) for viral entry into the cell. To achieve long-term control of viral replication by siRNA and prevent the emergence of escape variants, it is important to target highly conserved and/or essential HIV-1 sequences. For example, many sites in the cis-regulatory regions, as well as the protein-coding regions, of HIV-1 have been examined as potential targets for siRNA. These regions include the primer-binding site, the polypurine tract, the long terminal repeat, and the gag, pol, env, tat, rev, vif, and nef genes (6, 10, 11, 16, 18, 21, 30, 39). The degree to which siRNAs inhibited HIV-1 replication and the underlying mechanisms varied considerably, depending on the target sequence (10, 11). For example, RNAi-resistant HIV-1 variants can emerge not only through mutations in the siRNA target sequence but also through mutations that alter the local RNA structure (39). These results emphasize the need for empirical studies to determine effective siRNA target sites within the HIV-1 genome. In the present study, we selected several sequences for lentivirus-mediated shRNA expression based on a preliminary screening of HIV-1 RNAi target sites using synthetic siRNA duplexes. These sequences mapped within the integrase (IN) gene (int) and the attachment site (att), which are essential for HIV-1 integration. We evaluated the anti-HIV-1 activity of these expressed shRNAs using a highly susceptible CD4+ T-cell line. Genetic analysis of HIV-1 escape mutants that emerged after treatment with combinations of shRNAs revealed that two or more shRNAs targeting different essential sequences had the strongest impact on antiviral activity. The results also suggest that shRNAs or long-hairpin RNA (lhRNA) that targets incoming viral RNA before proviral DNA formation is more efficient at mediating RNAi antiviral therapy.

Retroviral integrase (IN) catalyzes the integration of viral cDNA into a host chromosome. Additional roles have been suggested for IN, including uncoating, reverse transcription, and nuclear import of the human immunodeficiency virus type 1 (HIV-1) genome. However, the underlying mechanism is largely unknown. Here, using a yeast two-hybrid system, we identified a survival motor neuron (SMN)-interacting protein 1 (Gemin2) that binds to HIV-1 IN. Reduction of Gemin2 with small interfering RNA duplexes (siGemin2) dramatically reduced HIV-1 infection in human primary monocyte-derived macrophages and also reduced viral cDNA synthesis. In contrast, siGemin2 did not affect HIV-1 expression from the integrated proviral DNA. Although Gemin2 was undetectable in cell-free viral particles, coimmunoprecipitation experiments using FLAG-tagged Gemin2 strongly suggested that Gemin2 interacts with the incoming viral genome through IN. Further experiments reducing SMN or other SMN-interacting proteins suggested that Gemin2 might act on HIV-1 either alone or with unknown proteins to facilitate efficient viral cDNA synthesis soon after infection. Thus, we provide the evidence for a novel host protein that binds to HIV-1 IN and facilitates viral cDNA synthesis and subsequent steps that precede integration in vivo.

We investigated the effects of signaling through CD28 family molecules on human immunodeficiency virus type 1 (HIV-1) replication in vitro. A monoclonal antibody (mAb) specific for inducible costimulator (ICOS) suppressed both X4 and R5 HIV-1 replication in CD4+ peripheral blood mononuclear cells (PBMC). This suppression was not attributable to reduced cell growth or viability. CD28 mAb showed variable effects and also suppressed HIV-1 replication when immobilized. Replication of pseudotype viruses with HIV-1-but not with vesicular stomatitis virus G-envelope was efficiently suppressed in CD4+ PBMC treated with ICOS or CD28 mAbs. However, CD4, CXCR4, and CCR5 expression on the surface was not down-regulated. Moreover, HIV-1 replication in CD4+ PBMC was suppressed by a soluble form of human B7-H2, a ligand of ICOS, but was enhanced by soluble B7-1, a ligand for CD28. These findings suggest that natural or artificial ligands for ICOS potentially suppress HIV-1 replication mainly at the entry stages.

Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia (ATL). Although the viral transactivation factor, Tax, has been known to have apparent transforming ability, the exact function of Tax in ATL development is still not clear. To understand the role of Tax in ATL development, we introduced short-interfering RNAs (siRNAs) against Tax in a rat HTLV-1-infected T-cell line. Our results demonstrated that expression of siRNA targeting Tax successfully downregulated Tax expression. Repression of Tax expression was associated with resistance of the HTLV-1-infected T cells to Tax-specific cytotoxic-T-lymphocyte killing. This may be due to the direct effect of decreased Tax expression, because the Tax siRNA did not alter the expression of MHC-I, CD80, or CD86. Furthermore, T cells with Tax downregulation appeared to lose the ability to develop tumors in T-cell-deficient nude rats, in which the parental HTLV-1-infected cells induce ATL-like lymphoproliferative disease. These results indicated the importance of Tax both for activating host immune response against the virus and for maintaining the growth ability of infected cells in vivo. Our results provide insights into the mechanisms how the host immune system can survey and inhibit the growth of HTLV-1-infected cells during the long latent period before the onset of ATL.

Previous studies have demonstrated that cyclopentenone prostaglandins (cyPGs) inhibit the replication of a wide variety of DNA and RNA viruses in different mammalian cell types. We investigated a new role for prostaglandin A1 (PGA1) in the inhibition of hepatitis C virus (HCV)-IRES-mediated translation. PGA1 exhibited dose-dependent inhibitory effects on HCV translation in HCV replicon cells. Furthermore, repetitive PGA1 treatment demonstrated the potential to safely induce the suppression of HCV translation. We also validated a new role for PGA1 in the inhibition of HCV-IRES-mediated translation by targeting cellular translation factors, including the small ribosomal subunit (40S) and eukaryotic initiation factors (eIFs). In pull-down assays, biotinylated PGA1 co-precipitated with the entire HCV IRES RNA/eIF3-40S subunit complex. Moreover, the interactions between PGA1 and the elongation factors and ribosomal subunit were dependent upon HCV IRES RNA binding, and the PGA1/HCV IRES RNA/eIF3-40S subunit complex inhibited HCV-IRES-mediated translation. The novel mechanism revealed in this study may aid in the search for more effective anti-HCV drugs.

Previous studies have demonstrated that cyclopentenone prostaglandins (cyPGs) inhibit human immunodeficiency virus type 1 (HIV-1) replication in various cell types. This antiviral activity has been associated with the induction of heat-shock protein 70 (HSP70) in infected cells. We investigated a new role of prostaglandin A₁ (PGA₁) in the replication of HIV-1 in non-permissive cells. Because overexpression of HSP70 blocks the viral infectivity factor (Vif)-mediated degradation of APOBEC3G (A3G) via the ubiquitin-proteasome pathway, we examined the effects of PGA₁ on A3G and HIV-1 replication. The induction of HSP70 synthesis by PGA₁ blocked Vif-mediated A3G degradation and enhanced the incorporation of A3G into both wild-type and Vif-deficient viruses. Furthermore, we determined the viral titer of HIV-1 particles produced from PGA₁-treated 293T cells. The induction of HSP70 synthesis by PGA₁ significantly reduced the viral titer in the presence of A3G. Additionally, the p24 Gag antigen levels were dramatically reduced in non-permissive cells treated once or repeatedly with PGA₁. Thus, we showed that PGA₁ inhibits HIV-1 replication, at least in part, by blocking Vif-mediated A3G degradation.

The human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) contains binding sites for several host transcription factors that contribute to HIV-1 gene expression. Although previous reports have indicated that HIV-1 Nef positively or negatively regulates HIV-1 gene expression, the precise molecular mechanisms by which this occurs remain largely unknown. In this study, we report that Nef suppressed LTR-driven transcription only in the presence of HIV-1 Tat, which was localized to the cytoplasm and degraded by the proteasome. However, the depletion of Hsp70 was found to reduce the suppressive effect of Nef on HIV-1 gene expression. These results suggest that Nef suppresses Hsp70-mediated HIV-1 Tat activation.Structured summary of protein interactionsTat and Nefcolocalize by fluorescence microscopy(View interaction)Hsp70physically interacts with Tat by anti tag coimmunoprecipitation(View interaction)